Sickel cell diagnostic test

ABSTRACT

A DIAGNOSTIC TEST AND REAGENT FOR THE RAPID IDENTIFICATION OF SICKLE-CELL ANEMIA AND SICKLE-CELL TRAIT COMPRISING REACTION OF UNCLOTTED BLOOD WITH AN ADMIXTURE OF (NH4)2SO4/K2HPO4 BUFFER, PH 7-7.5, SAPONIN AND SODIUM DITHIONITE AND OBSERVATION OF THE TURBIDOMETRICCOLORMETRIC CHARACTERISTICS.

United States Patent US. Cl. 23-230 B 7 Claims ABSTRACT OF THEDISCLOSURE A diagnostic test and reagent for the rapid identification ofsickle-cell anemia and sickle-cell trait comprising reaction ofunclotted blood with an admixture of (NH SO /K HPO buffer, pH 7-7.5,saponin and sodium dithionite and observation of theturbidometriccolormetric characteristics.

This invention relates to a diagnostic test and reagent. Moreparticularly, this invention relates to a method for the rapididentification of sickle-cell anemia and sicklecell trait.

The term sickle-cell disease refers to conditions and diseasescharacterized by the presence of hemoglobin S (HbS). Hemoglobin S is aninherited characteristic and is probably the most widely encounteredhemoglobin variant in the United States. IWhen present in theindividual, hemoglobin S is either homozygous (SSsicklecell anemia) orheterozygous (AS-sickle-cell trait). It can also be found in combinationwith other abnormal hemoglobins and with other hereditary diseases. Theimportance of adequate diagnosis of sickle-cell disease lies in the factthat if unrecognized and/or untreated, the disease may be fatal. Undercertain conditions that cause low oxygen tension, sickle-cell trait canresult in serious and even fatal clinical complications. Sickle-cellanemia may prove fatal before adolescence but with appropriate medicalcare it is possible to extend survival for 30 or 40 years.

Various methods of diagnosis of sickle-cell disease have been reportedheretofore. They include electrophoresis, differential solubility testsand slide elution tests. Of these, the original differential solubilitytest of Itano, I. of Haematology, 4, pp. 66-68 (1949), Arch. Biochem.Biophys, 47, pp. 148-59 (1953), Science, 117, pp. 89-94 (1953), andmodifications thereof are most prevalent in use. One such modificationdescribed in US. Pat. 3,492,- 095 involves the steps of adding sodiumdithionite reductant to a high ionic concentration phosphate buttersystem, adding a saponin hemolyzing agent thereto and then adding agiven amount of the blood to be tested. After mixing and standing for aperiod of time, the resultant solution is observed for translucense orturbidity. Turbidity indicates the presence of hemoglobin S whereas ifthe solution remains translucent, the results indicate the absence ofhemoglobin S.

While the foregoing modification is useful for determining the absenceor the probable presence of hemoglobin S, it does not provide fordifierentiation between sickle-cell anemia (SS) and sickle-cell trait(AS).

Accordingly, it is an object of this invention to provide a diagnostictest and reagent for the determination of hemoglobin S.

It is another object of this invention to provide a diagnostic test andreagent for the rapid identification of sickle-cell anemia andsickle-cell trait.

Other objects and advantages of the present invention will be apparentto those skilled in the art after reading the disclosure hereof.

In brief, the present invention resides in the provision of a new buffersystem and a macroscopic turbidometricice colorimetric procedure fordistinguishing between sicklecell anemia and sickle-cell trait.

According to past practice it has been customary to use a phosphatebutter of high ionic concentration. Thus, US. Pat. 3,492,095 describesthe use of a buffer containing 16.9 grams of KH2PO4 and 21.7 grams of KHPO (or a total of 38.6 grams of phosphate salts) in ml. of water andhaving a high hydrogen ion concentration and concomitant pH of 6.5 to6.8. In the present invention the new buffer system comprisesessentially (NH SO of relatively low ionic concentration and only asmall amount of K HPO The (NH,) SO which at normal room temperature(2025 C.) forms a 76% saturated solution in water, is used at aboutone-third saturation and preferably at a level of from about 21 to about35 grams per 100 ml. Adrnixed with the (NH SO to form the present bufferis about 0.5 to about 2 grams of K2HPO4 per 100 ml. Thus, the overallbuffer solution has a relatively low ionic concentration in terms of thesaturation point and only a small fraction thereof comprises phosphate.Instead of having a high hydrogen ion concentration, a relatively lowhydrogen ion concentration is employed in the buffer of the presentinvention whereby the pH is neutral to slightly alkaline and preferablyfrom about 7 to about 7.5. For example, a representative buffer of thepresent invention having a pH of 7.1 has only one-fourth A) the numberof hydrogen ions that are in a buffer having a pH of 6.5.

In addition to the new buffer system described herein, the reagentsemployed in the present invention include a red cell hemolytic agentsuch as, for example, saponin and a reducing agent such as, for example,sodium dithionite. The use of saponin as a rapid hemolytic agent formeasuring hemoglobin content has been disclosed heretofore in US. Pat.2,519,999, col. 51, lines 2-10, and the use of sodium dithionite as areducing agent in a test for hemoglobin has been described previously byItano and Pauling, J. of Haematology, 4, pp. 66-68 (1949). However, ithas not been known heretofore to use the latter two reagents incombination with the new buifer system defined herein. By employingthese three reagents together it has now been made possible to obtain aconvenient and rapid identification of sickle-cell anemia andsickle-cell trait in one test whereas previously it was possible only toobtain identification of hemoglobin S with the high ionic concentrationphosphate buffer, saponin, and sodium dithionite. In said prior test ithad been necessary to conduct a separate and cumbersome electrophoresistest to differentiate between sickle-cell anemia and sickle-cell trait,thereby disadvantageously requiring a second visit to the clinicallaboratory by all those patients whose initial test was found to bepositive for hemoglobin S. Health Services and Mental HealthAdministration Health Reports, 87 (1), pp. 9-12 (1972).

In accordance with the method of this invention, the hemolytic agent ismixed with the buffer and then the reductant is admixed therewith. Asample of test blood such as, for example, from a finger or heelpuncture or anticoagulated venous blood is then mixed with a suitablealiquot of the resultant reagent mixture in a micro test tube and thesolution allowed to stand at normal room temperature for severalminutes, e.g., about five minutes, after which the test tube is viewedin the path of a fluorescent light with a metal reflector to determinewhether the solution is translucent or turbid. If the solution istransulcent, the hemoglobin is AA (normal), whereas if the solution istubid, then the hemoglobin is AS (sickle-cell trait) or SS (sickle-cellanemia).

A further differential of the gene types can then be made in the sametest by spinning the test tube with the turbid sample in a serologicalcentrifuge for several minutes, e.g., about three minutes, at about 3000to 5000 r.p.m. At the end of this time period the test tube is viewedagain for color characteristics in the fluorescent light path.

Hemoglobin S is insoluble in the new buffer system of this invention andthe SS hemoglobin is a yellow solution with red precipitate. The AShemoglobin is a pink solution with a red precipitate. Normal AAhemoglobin is soluble in the new buffer system of this invention withonly a very small amount of a white precipitate.

The method of this invention has been found to give a 100% correlationbetween the S gene and hemoglobin electrophoresis. It should beunderstood, however, that the method does not differentiate the betathalassemia (Th) or C gene and there is a potential error or two personsout of 100 being diagnosed as AS when they could be CS or ThS. There isa potential error of 3.8% in diagnosis as AA when the patient may havean additional hemoglobin problem of Th or a C gene.

It is thus seen that the diagnostic test and reagent of the presentinvention provides for the rapid identification of sickle-cell anemiaand sickle-cell trait in one test with only a small recognizable marginof error in the diagnosis of other hemoglobinopathies.

A further advantage of the present invention is that the differentiationof sickle-cell anemia and sickle-cell trait is carried out byobservation of a sharply defined precipitate which rapidly settles atthe bottom of the test tube and a distinct color difference in thesupernantant solution. Differentiation between sickle-cell trait andsicklecell anemia has been disclosed heretofore by Huntsman et al., J.Clin. Path., 23, pp. 78183 (1970), but in the method described thereinthe turbid material rises to the surface of the solution. This causessignificant problems due to the inability to obtain complete separation,the necessity of spinning in a hematocrit centrifuge, and the tendencyof the entire precipitate to drop from the surface during thecentrfuging or braking of the centrifuge and in the normal handling ofthe test tubes in the laboratory thereafter. This fragility of theturbid material at the top of the test tube whereby it falls into thesolution tends to give false readings of the sickle-cell diagnosis.

The transillumination of the micro test tube containing the sickle-celldiagnostic reagent and the test blood sample can be conveniently carriedout in a fluorescent illuminator such as described in the co-pendingutility application of Seitz and Miranda, Ser. No. 237,824, filedconcurrently herewith and assigned to a common assignee. A micro testtube, 12 mm. diameter x 75 mm. long, can have a dark letter A, about -10mm. high, printed on the side of the tube opposite the viewer butadjacent the illuminator. So long as the solution remains translucent,the letter A can be read through the tube, but when the solution becomesturbid, the letter A can no longer be seen through the tube.

In accordance with a preferred embodiment of the invention, the newbuffer system is provided together with the other reagents in a completekit form for the diagnostic test. The buffer can be in liquid form butit is preferred to provide the hemolytic agent and reductant in a dry,powdered form to extend the shelf life of the test kit. In particular,the reductant sodium dithionate tends to lose S0 in aqueous solutionover a period of time.

Prior to use, the powdered hemolytic agent and the powdered reductant oran admixture thereof can be admixed with the buifer solution and theresulting solution can then be used to conduct a plurality of diagnostictests for sickle-cell disease. Thus, 0.55 grams each of saponin andsodium dithionite in 55 ml. of buffer solution is sufficient for aboutsickle-cell diagnostic tests.

According to yet another embodiment of the invention, the buffersolution contains a re-dox indicator dye such as, for example, methyleneblue, which will change color by virtue of the break-down of the sodiumdithionite reductant during storage when admixed with the aqueous buffersolution. Thus, methylene blue is colorless in the reduced state andblue in the oxidized state. So long as the buffer containing the sodiumdithionite remains colorless, or yellow due to the pressure of thesaponin hemolytic agent, the reagent system is suitable for carrying outthe diagnostic test of this invention. At such time as the buffer systemturns blue due to the break-down of the sodium dithionite during storageand conversion of the methylene blue to the oxidized state, the reagentshould be discarded.

Another suitable re-dox indicator dye system which can be used in thisinvention is basic fuchsin, which is violet red in oxidized form andcolorless in reduced form.

The diagnostic test and reagent of this invention also lends, itselfwell for use on automated analytical equipment. For example, it can beadapted for application on the Autoanalyzer AAII instrument manufacturedby Technicon Corp. and on various single channel instruments.

The following examples will further illustrate the present inventionalthough it will be understood that the invention is not limited tothese specific examples.

EXAMPLE 1 A complete sickle-cell diagnostic kit is provided with onehundred (12 mm. x 75 mm.) micro test tubes, each being calibrated at 2ml. and having a dark letter A, 6 mm. high, printed on the outside; twobottles, each containing 105 ml. of a special buffer; two bottles, eachcontaining 1.1 grams of dry, powdered saponin; and two bottles, eachcontaining 1.1 grams of dry, powdered sodium dithionite. This kit isadapted for carrying out sickle-cell diagnostic tests.

The special butter is prepared as follows: a 28% solution of (NH SO ismade-up by dissolving 280 grams of the sulfate salt in one liter ofdistilled water. A molar solution of K HPO is made-up by dissolving 174grams of the phosphate salt in one liter of distilled water. To oneliter of the (NH SO solution is then added 60 ml. of the K HPO solution.The pH of the resulting buffer is 7.1.

One unit of 1.1 gram of saponin is mixed with one unit of ml. of buffersolution and then one unit of 1.1 gram of sodium dithionate is admixedtherewith. The resulting solution is stable for about 30 days at about2"- 8 C. and is suitable for carrying out the diagnostic method of thisinvention for the rapid identification of sickle-cell anemia andsickle-cell trait.

EXAMPLE 2 Diagnosis for sickle-cell disease by use of the final reagentsolution for Example 1, above, is carried out as follows:

An aliquot comprising 2 ml. of the reagent solution is pipetted into oneof the micro test tubes. A one-tenth A ml. sample of the patients blood,which is nonclotted, is then mixed into the solution and the mixtureallowed to stand at room temperature for five minutes. After the fiveminute setting period, the test tube and reagen solution is viewed in afluorescent illuminator to determine whether the solution remainstranslucent (HbA) or becomes turbid (HbS) The test tube and reagentsolution is then spun in a serological centrifuge for three minutes at3500 rpm. and again viewed in the fluorescent illuminator to determinewhether the solution is translucent (HbAA), or pink with a redprecipitate (HbAS), or yellow with a red precipitate (HbSS).

The above differentiation between sickle-cell anemia (HbSS) andsickle-cell trait (HbAS) is 97% reliable with respect to allhemoglobinopathies and the determination of the sickle-cell gene (S) is100% reliable. The entire test is conducted in only about 8 minutes,thereby providing a rapid diagnosis of the patient.

Although specific amounts and proportions of hemolytic agent andreductant are described in the foregoing examples, it will be apparentthat variations from these examples can be made in the diagnostic testand reagent of this invention without departing from the basic and novelconcepts thereof. In general, these materials are each used withinranges of from about 0.01 to about 2 grams per 100 ml. of solution.Conventional hemolytic agents and reductants other than thosespecifically described herein can also be used with substantiallyequivalent results.

Various other examples and modifications of the foregoing examples willbe apparent to the person skilled in the art after reading the abovedisclosure and the appended claims without departing from the spirit andscope of the invention. All such further examples and modifications areincluded within the scope of said claims.

What is claimed is:

1. A reagent for the diagnosis of sickle-cell disease comprising amixture of an aqueous bufi'er solution, a hemolyzing agent and areducing agent, said buffer comprising from about 21 to about 35 gramsof (NH S0 per 100 ml. and from about 0.5 to about 2.0 grams of K HPO per100 ml. and having a pH of from about 7 to about 7.5.

2. The reagent of claim 1 in which the buffer contains about 28 grams of(NH SO per 100 ml. and about one gram of K HPO per 100 ml.

3. The reagent of claim 1 in which the hemolyzing agent is saponin.

4. The reagent of claim 1 in which the reducing agent is sodiumdithionite.

5. The reagent of claim 1 in which the mixture contains about 28 gramsof (NH SO about one gram of K HPO about one gram of saponin, and aboutone gram of sodium dithionate per 100 ml. of solution.

6. A method for the rapid dilferentiation of sickle-cell anemia andsickle-cell trait comprising admixing a sample of unclotted blood with apredetermined amount of the reagent of claim 1 and observingmacroscopically for turbidity in the solution as indicative of presenceof HbS, then centrifuging and observing macroscopically for colorcharacteristics in the solution in which a pink solution is indicativeof sickle-cell trait and a yellow solution is indicative of sickle-cellanemia.

7. The method of claim 6 in which blood sample and reagent are admixedin proportions of about 100 microliters of blood and about 2 millilitersof reagent.

US. Cl. X.R. 252-408

